Primary Motility  Disorders of the  Esophagus
 The Esophageal
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 Esophagogastric  Junction
 Barrett's
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OESO©2015
 
Volume: Barrett's Esophagus
Chapter: Diagnosis
 

Are chromosomal alterations observed in non-dysplastic Barrett's mucosa

C.K. MacKay, R.C. Stuart (Glasgow)

Cancer development in Barrett's esophagus is a multistage process, where an environmental stimulus (e.g. acid and/or bile) and a genetic predisposition result in genomic instability. Cells with multiple genetic abnormalities develop, resulting in clones with accumulating genetic errors. Some clones will gain a proliferative advantage, and one may develop the capacity for invasion, resulting in a cancer [1]. This theory of polyclonal evolution may account for the histological heterogeneity found in Barrett's esophagus [2] and the variable natural history of dysplastic progression [3].

Genetic instability and clonal evolution have been demonstrated in Barrett's esophagus, using flow cytometry which has described ploidy patterns in the progression to cancer [4, 5]. The presence of such aneuploid peaks are due to increased or abnormal DNA content, after structural and numerical chromosomal alterations, that are associated with increasing genomic instability. The prevalence of such aneuploid populations increases with increasing histological risk of malignancy and multiple aneuploidies are usually associated with high-grade dysplasia or cancer [6].

There are many studies demonstrating such changes in esophageal adenocarcinomas and the surrounding metaplastic epithelium [3, 4, 6], but the information on truly preneoplastic mucosa in patients with no dysplasia or cancer is sparse. Finding changes in such mucosa is important, as it implies genomic instability before cancer develops and the detection of early events increases our understanding of the order of malignant progression.

The presence of multiple aneuploid populations implies the presence of clones with abnormal DNA content, but as part of the flow cytometry process, the tissue is broken down, and mucosal heterogeneity is lost. The mucosal cells are also contaminated by stromal cells, with no malignant potential after tissue integrity is lost. Furthermore different clones within the same specimen are analysed simultaneously and the results are grouped together. Identification of chromosomal abnormalities and correlation with histology requires the maintenance of tissue integrity and can be performed using interphase cytogenetics. This allows the genetic profile of different cells within the metaplastic segment to be examined for separate clonal populations, with different chromosomal rearrangements.

Whilst the maintenance of tissue integrity is vital to relate the complex, heterogeneous histological and cytogenetic profile in Barrett's esophagus, there are features specific to the condition and the esophagus that cause difficulties with such a process. Tissue microdissection relies on the existence of paraffin embedded tissue of appropriate size, which is mainly available in esophagectomy specimens, where surgery has been performed for cancer or high-grade dysplasia. Analysis of such specimens will show a higher number of abnormal chromosomal profiles, but any changes identified cannot be defined as early. Patients with uncomplicated disease only undergo surveillance with endoscopic biopsies, which must be frozen if interphase cytogenetics are to be performed and are too small to allow tissue microdissection [7]. Furthermore changes are less likely to be frequent in truly benign disease, and the complex histological pattern of Barrett's esophagus can make it difficult to relate such changes to the histology.

The majority of work on chromosomal alterations in Barrett's esophagus has focused on changes that are discovered in cancers and the surrounding metaplastic epithelium [8-10]. Structural changes have been identified in chromosome 1p, 3q,11p and 22p in dysplastic mucosa or adenocarcinomas [11, 12]. The progression to carcinoma appears to be associated with allelic losses at sites of tumour suppressor genes [13], such as the p53 gene, located on chromosome 17p. Allelic losses at this site have been identified in increasing frequency as malignant potential develops [14, 15], but have also been found in diploid populations [14], implying that this may be an early event. Further losses of heterozygosity have been identified on chromosomes 5q, 8p, 13q, 18q [8, 13] in metaplastic tissue surrounding cancers.

Numerical chromosomal abnormalities have also been described in dysplastic mucosa and adenocarcinomas, with the most frequent abnormality being loss of the Y chromosome, although losses of chromosomes 4, 18 and 21 have been identified [11, 12]. Very little is known on karyotypic abnormalities in patients with uncomplicated preneoplastic Barrett's esophagus, although Raskind [16] did identify cytogenetically abnormal clones (chr.Y loss & chr. 7 gain) on endoscopic biopsies, in patients that later progressed to develop dysplasia or cancer. Further evidence of early chromosomal changes is recent work showing microsatellite instability in endoscopic biopsies taken from areas of uncomplicated intestinal metaplasia [17, 18].

We have recently searched for numerical chromosomal alterations in preneoplastic Barrett's esophagus [19], by performing interphase cytogenetics using fluorescent-in situ hybridisation (FISH) on frozen biopsies from multiple levels of the metaplastic segment in 20 patients. FISH entails hybridisation of chromosomes with centromere specific probes, which are then labelled with a fluorochrome, allowing visualisation of numerical chromosomal abnormalities while retaining tissue integrity. Chromosome specific probes to 1, 8, 9, 12, 17 and 18 were used and the normal chromosome index was calculated by analysing sections from normal gastric and squamous esophageal biopsies, and dividing the total number of signals counted by the number of nuclei counted. As tissue integrity is maintained, it is possible to count only mucosal cells, avoiding "contamination", by excluding the stromal cells from analysis. A value above or below 3 standard deviations of this value (1.55) would be abnormal, when the same analysis and calculation was applied to the sections from the Barrett's segment, and represents gain or loss respectively of a particular chromosome. Two hundred and fifteen sections from Barrett's metaplasia sections were examined and 7(3%) showed numerical chromosomal alterations were identified (Table I).

Table I. Changes in chromosome copy number in study population according to patient and biopsy site - biopsies from squamo-columnar junction (Z-line), or Barrett's levels (B1, B2 & B3).

This low percentage of karyotypic abnormalities is expected as genomic instability is rare in truly benign disease, and none of these patients had dysplasia. Interestingly, this figure is similar to the percentage of patients showing aneuploidy in non-dysplastic mucosa. One patient had a gain of chromosome 18 (Figure 1) at 2 different levels within the metaplastic segment, suggesting a field change phenomenon, with a clone of abnormal cells extending throughout the mucosa. Another patient had gains of both chr. 8 and 9 in biopsies from different levels of the Barrett's segment, which supports the theory that the disease has a polyclonal nature.

Alterations in chromosome structure and number can be identified in non-dysplastic Barrett's esophagus, although these events are rare. The prevalence of such changes does appear to increase along with increasing malignant potential and correlates with the degree of dysplastic progression. Unfortunately our understanding of this process of malignant transformation is far from complete and the variable natural progression of dysplasia continues to cause problems in surveillance programmes. Whilst it is unlikely that the discovery of early chromosomal alterations will help us perform cancer surveillance, it

Figure 1. A. Normal hybridisation in a Barrett's section using a centromeric probe to chr. 18. Note that most nuclei show only 2 signals from chromosomes. B. Barrett's section showing gain of chr. 18. Note no. of nuclei showing > 2 chromosomes compared with A.

lends evidence to the early process of genomic instability, and may be vital in our eventual understanding of this carcinogenesis.

References

1. Blount PL, Rabinovitch PS, Haggitt RC, Reid BJ. Early Barrett's adenocarcinoma arises within a single aneuploid population. Gastroenterology 1990;98:A273.

2. Paull A, Trier JS, Dalton MD. The histologic spectrum of Barrett's esophagus. N Engl J Med 1976;295:476-480.

3. Reid BJ, Blount PL, Rubin CE, Levine DS, Haggitt RC, Rabinovitch PS. Flow-cytometric and histological progression to malignancy in Barrett's esophagus:prospective endoscopic surveillance of a cohort. Gastroenterology 1992;102:1212-1219.

4. Reid BJ, Haggitt RC, Rubin CE, Rabinovitch PS. Barrett's esophagus. Correlation between flow cytometry and histology in detection of patients at risk for adenocarcinoma. Gastroenterology 1987;93:1-11.

5. Fennerty MB, Sampliner RE, Way D, Riddell RH, Steinbronn K, GarewalHS. Discordance between flow cytometric abnormalities and dysplasia in Barrett's esophagus. Gastroenterology 1989;97:815-820.

6. Rabinovitch PS, Reid BJ, Haggitt RC, Norwood TH, Rubin CE. Progression to cancer in Barrett's esophagus is associated with genomic instability. Lab Invest 1989;60:65-71.

7. Reid BJ, Haggitt RC, Rubin CE, et al. Observer variation in the diagnosis of dysplasia in Barrett's esophagus. Hum Pathol 1988;19:166-178.

8. Dolan K, Garde J, Walker SJ, Sutton R, Gosney J, Field JK. LOH at the sites of the DCC, APC, and TP53 tumor suppressor genes occurs in Barrett's metaplasia and dysplasia adjacent to adenocarcinoma of the esophagus. Hum Pathol 1999;30:1508-1514.

9. Barrett MT, Sanchez CA, Prevo LJ, et al. Evolution of neoplastic cell lineages in Barrett's oesophagus. Nat Genet 1999;22:106-109.

10. Wu TT, Watanabe T, Heitmiller R, Zahurak M, Forastiere AA, Hamilton SR. Genetic alterations in Barrett's esophagus and adenocarcinomas of the esophagus and esophagogastric junction region. Am J Pathol 1998;153:287-294.

11. Rodriguez E, Rao PH, Ladanyi M, et al. 11p13-15 is a specific region of chromosome rearrangement in gastric and esophageal adenocarcinomas. Cancer Res 1990;50:6410-6416.

12. Menke-Pluymers MB, Van Drunen E, Vissers KJ, Mulder AH, Tilanus HW, Hagemeijer A. Cytogenetic analysis of Barrett's mucosa and adenocarcinoma of the distal esophagus and cardia. Cancer Genet Cytogenet 1996;90:109-117.

13. Huang Y, Boynton RF, Blount PL. Loss of heterozygosity involves multiple tumor suppressor genes in human esophageal cancers. Cancer Res 1992;52:6525-6530.

14. Blount PL, Ramel S, Raskind WH, et al. 17p allelic deletions and p53 protein overexpression in Barrett's adenocarcinoma. Cancer Res 1991;51:5482-5486.

15. Blount PL, Meltzer SJ, Yin J, Huang Y, Krasna MJ, Reid BJ. Clonal ordering of 17p and 5q allelic losses in Barrett dysplasia and adenocarcinoma. Proc Natl Acad Sci USA 1993;90:3221-3225.

16. Raskind WH, Norwood T, Levine DS, Haggitt RC, Rabinovitch PS, Reid BJ. Persistent clonal areas and clonal expansion in Barrett's esophagus. Cancer Res 1992;52:2946-2950.

17. Meltzer SJ, Yin J, Manin B, et al. Microsatellite instability occurs frequently and in both diploid and aneuploid cell populations of Barrett's-associated esophageal adenocarcinomas. Cancer Res 1994;54:3379-3382.

18. Gleeson CM, McDougall NI, Russel SE, McGuigan JA, Collins JS, Sloan JM. Microsatellite analysis provides evidence of neoplastic transformation in long-segment, but not in short-segment Barrett's oesophagus. Int J Cancer 2000;85:482-485.

19. MacKay CK, Stuart RC, Going J, Baxter JN, Keith WN. Interphase cytogenetics of non-dysplastic Barrett's esophagus. Gastroenterology 1997;112:A607.


Publication date: August 2003 OESO©2015